CN112028980A

CN112028980A - Sericin, extraction method, application of sericin as cellular antiviral immunopotentiator and application of sericin

Info

Publication number: CN112028980A
Application number: CN202010961734.1A
Authority: CN
Inventors: 吴建国; 李永奎; 黄颖; 邢曦雯; 罗震; 潘攀; 张业顺
Original assignee: Jinan University
Current assignee: Jinan University
Priority date: 2020-09-14
Filing date: 2020-09-14
Publication date: 2020-12-04
Anticipated expiration: 2040-09-14
Also published as: CN112028980B

Abstract

The invention belongs to the technical field of extraction of natural protein components and virology, and discloses sericin, an extraction method, application of the sericin as a cellular antiviral immunopotentiator and application of the sericin, and application of the sericin in preparation of a medicament for preventing and controlling viral infection; the dose concentration of sericin is 10-20mg/mL, and the effective final concentration is more than or equal to 1 mg/mL; the extraction method of sericin comprises the following steps: selecting available silkworm cocoons and storage conditions, pretreating and sterilizing the silkworm cocoons, dissolving sericin, dialyzing, renaturing sericin, concentrating a sericin solution, removing endotoxin in the sericin solution, filtering the sericin solution, controlling and detecting the quality of the sericin solution, and taking the sericin solution which meets the detection standard as a cellular antiviral immunopotentiator. The invention develops and utilizes the fibroin sericin for the first time, and the fibroin sericin is used for preventing and controlling the virus infection. The invention has wide raw material sources, and low cost compared with the high synthesis and production cost of other antiviral medicaments.

Description

Sericin, extraction method, application of sericin as cellular antiviral immunopotentiator and application of sericin

Technical Field

The invention belongs to the technical field of extraction of natural protein components and virology, and particularly relates to sericin, an extraction method, a cellular antiviral immunopotentiator and application.

Background

Currently, sericin is a natural protein existing in silkworm cocoons, and is an active ingredient coated on the outer layer of silk. In the textile industry, silk cocoons undergo a spinning process to extract silk fibroin used to make textiles, and sericin has previously been generally discarded as industrial waste. In recent years, with the development of bioengineering and materials, researchers have gradually discovered the use value, especially the discovery that the substance has multiple biological activities, the substance becomes a research hotspot, and the change of waste into valuable becomes possible. Many scientific researches show that the sericin has the activities of low immunogenicity, promoting cell adhesion and proliferation activity, promoting metabolism, promoting cell activity and the like, can be used as an active ingredient in high-grade cosmetics, and is also developed and researched in the fields of food and medical treatment.

The traditional sericin extraction process mainly adopts a water boiling method, which dissolves sericin components in silkworm cocoons by heating and then collects the components dissolved in an aqueous solution. Has the advantages of convenient method and low cost. The disadvantages are that: 1. the extracted sericin has more impurities and may be mixed with a large amount of other components of fibroin and silkworm cocoons; 2. sericin is very unstable in a heated aqueous solution and is easy to degrade, and most of the sericin is a degradation product of sericin; 3. furthermore, the time is long, since the dissolution of sericin in hot water is time-consuming. Reducing time can reduce sericin degradation, but yields are greatly compromised, making it difficult to meet both high yield and product integrity. The sericin obtained by the method is low in quality and purity, and can be used only for feed or industrial raw materials, such as raw materials for producing amino acid.

The alkali dissolution method is a technique which is an improvement of the conventional method. The sericin is more easily dissolved in a weak alkaline solution, the dissolving speed of the sericin is greatly accelerated, and the quality and the yield of the obtained sericin are improved to a certain extent compared with the traditional method by heating at a lower temperature. Nevertheless, the quality and purity of sericin obtained by this method can never satisfy biomedical research and application requirements.

The lithium bromide method is an effective novel sericin extraction method, and mainly adopts a high-concentration lithium bromide solution to dissolve sericin at room temperature. The method can obtain complete sericin molecules with high yield. Lithium ions and bromide ions dissolved in the sericin solution are difficult to completely remove, so that the obtained sericin is difficult to apply to the biomedical field.

The urea method is also a new method for effectively extracting sericin, which is to denature and dissolve sericin in silkworm cocoons by using a high-concentration urea solution and then obtain high-purity complete sericin through renaturation. The method has the advantages that the obtained sericin has high purity and integrity, and the used dissolving medium has low toxicity and is easy to remove. The disadvantage is that sericin goes through the process of denaturation and renaturation in the urea method, and the high-grade structure of the finally obtained protein may be different from the natural structure.

Sericin is a protein molecule which is synthesized and secreted by three genes, Sericin-like 1, Ser2 and Ser3, encoded by silk gland cells of silkworms. The amino acid sequence of sericin comprises a plurality of serine-rich repeats, such that the percentage of serine is up to 42.1%, and a carbinol group (-CH2OH) consisting of one secondary carbon atom (-CH2-, one C atom connecting 2H atoms) and one alcoholic hydroxyl group is present on each serine residue in the polypeptide chain. The homoserine character of sericin determines that the protein sequence is rich in carbinol (-CH2OH) or alcoholic hydroxyl (-OH) groups. The organic compound rich in alcoholic hydroxyl groups has good water absorption, and sericin also has good water absorption, so sericin is developed as a skin care product, namely, the activity of moisturizing the skin by water absorption is utilized. The carbinol group (-CH2OH) is easily oxidized to aldehyde group (-CHO), and further oxidized to carboxyl group (-COOH), and has reducibility. Therefore, sericin rich in methanol groups (-CH2OH) has reduction activity and is a good biological antioxidant, and skin care products developed by the sericin have the functions and effects of resisting oxidation and aging.

The water absorption and antioxidant activity are determined by the chemical composition of sericin, and are important chemical activities of sericin. The two chemical activities of sericin are well researched and analyzed, and the sericin has a certain application. However, in addition to this, sericin is a biological macromolecular protein, and its biological activity is more worthy of study, namely, whether sericin can regulate the molecular signaling and the reaction process of living macromolecules of cells, tissues, organs and individuals? What regulatory effect on cellular function? And what valuable biological activity or function is sericin? Some researchers have found a lot of excellent biological activities of sericin, sericin can promote the proliferation of certain cells, promote the repair of damaged tissues, inhibit inflammatory reaction, and crosslinked sericin hydrogel has cell adhesion. Also, it has been found that sericin has certain antitumor activity. These studies provide good insights into the development and utilization of sericin. However, sericin, a natural biomaterial, is developed and applied to cells and human bodies for specific activities, and particularly, the biological safety and biological activity of the biomaterial need to be considered.

The virus is always an unconscious demon, and the search for an effective method for the virus is an object which is always cumin by virology researchers. In fact, human cells have a natural mechanism for preventing and controlling viruses, namely the natural immune response system of the cells, which is the first line of defense of human against viral infection. When the virus infects cells, the cells have a plurality of sensors to capture certain components of the virus, and after capture, the sensors can activate downstream cell signals to mediate the cells to start expressing interferon, and the interferon can induce a plurality of antiviral mechanisms of the cells. Intracellular RIG-I, Mda5 and TLR3/7, for example, recognize specific components of the virus, activating interferon expression, and thereby initiating a downstream cellular antiviral response. Most of the current antiviral drugs are screened or designed mainly aiming at the processes of adsorption, invasion, expression, replication and the like of viruses. The existing immunopotentiators mainly play a role by improving the innate immunity and the acquired immunity of the body. The sericin provided by the invention can specifically improve the activation of a mode receptor for recognizing virus RNA and downstream signals thereof in cells, thereby enhancing the sensitivity of the cells to viruses, activating interferon more quickly and generating stronger antiviral immune response.

Through the above analysis, the problems and defects of the prior art are as follows: (1) the sericin product obtained by the existing sericin extraction method has problems in the aspects of biological safety, structural integrity, biological activity, higher product yield and the like, and can not meet the conditions at the same time.

(2) The prior art does not provide effective technical support for the application of active sericin in antivirus.

(3) The existing immunopotentiators mainly enhance the innate immunity and the acquired immunity of organisms, have long response time to virus infection, are not used for enhancing the recognition of cells to viruses and promoting the antiviral response of infected cells, and have weak pertinence and specificity.

The difficulty in solving the above problems and defects is: (1) the difficulty in solving the biological safety of the extracted sericin lies in: sericin is a natural component of silkworm cocoon, raw materials can adsorb various microorganisms and impurities from the environment, and the common extraction method is difficult to avoid the pollution of the microorganisms and harmful substances in the extraction process. A set of scientific and reasonable extraction method and detection scheme is established, the technical scheme comprises the sterilization treatment of raw materials, the sterilization of used reagents, devices and environment, the dialysis and purification of primary products, the detection of heat source substances and the like, and the microorganisms and harmful substances are prevented from being mixed into the sericin product on the premise of ensuring the structural integrity and the product yield of the sericin.

(2) Existing extraction methods can alter the native conformation or destroy the structural integrity of sericin, which can seriously impair the biological activity of sericin products. To maximally dissolve sericin from silkworm cocoon, a compound which changes the protein conformation such as urea, LiBr, alkali and the like, or a heating method which breaks the peptide chain of protein, urea, LiBr, heat and the like which changes the spatial conformation of the peptide chain of protein is used. Avoids selecting alkali and high temperature, selects milder conditions for dissolution and extraction, can avoid the sericin molecules from being damaged, but is difficult to maintain the natural conformation of the sericin molecules. In addition, how to select the silkworm cocoon with the highest sericin biological activity as a raw material is also a key ring.

(3) The dissolving property of the sericin determines that a sericin solution product has high enough concentration, can keep a natural structure complete, does not contain urea, LiBr and the like as denaturants with dissolution promotion effect, and is a technical problem which is difficult to solve. In the solution of urea and LiBr, the spatial conformation of the peptide chain of sericin is changed, the solubility is very high, and the polypeptide chain composing sericin can be refolded if small molecules such as urea or LiBr are removed by dialysis and the like. Practice shows that after the concentration of urea or LiBr is gradually reduced, the sericin with high concentration can be irreversibly precipitated, which indicates that the solubility of the sericin is reduced due to the fact that the polypeptide chain of the sericin is subjected to misfolding. Molecules such as urea and LiBr are components that must be removed as sericin solutions for cellular and in vivo applications. Furthermore, a sufficiently high sericin concentration, with intact natural structure, is necessary for the action of cellular antiviral immunopotentiators.

(4) The immunopotentiator for resisting virus infection is required to have no toxicity, no harm and high biological safety, and also has a specific enhancement effect on cell antiviral reaction. Cellular molecules and signaling pathways involved in the innate immune response of cells against viruses are complex, but follow rigorous molecular mechanisms. Among natural nuisanceless substances, it is also an inventive task to find substances that specifically enhance the cellular antiviral innate immune response.

The significance of solving the problems and the defects is as follows: solves the problem of biological safety of sericin extraction, is a precondition that the natural material can be applied to cells, animal models and human bodies,

maintaining the structural integrity of sericin is essential for sericin to exert biological activity.

A sufficiently high concentration of active sericin solution to be of biomedical use.

Natural and harmless substances are searched and developed to be used as a specific cellular antiviral immunopotentiator, so that the high efficiency and practicability of the immune effect enhancement can be ensured, and the safety is also ensured.

Disclosure of Invention

Aiming at the problems in the prior art, the invention provides sericin, an extraction method, a cellular antiviral immunopotentiator and application. In particular to the extraction of active sericin from silk and an antiviral test aiming at various viruses, especially the combination of virology technology and material science technology. The natural sericin extracted by the invention can enhance the natural immunoreaction capability of cells under certain conditions, thereby protecting the cells from virus infection.

The invention aims to extract active sericin with high biological safety and complete structure from silkworm cocoons, and firstly proposes the application of the natural material in resisting virus. The sericin provided by the invention has high purity, no endotoxin, complete structure and biological activity, and can be directly used for cell virus infection models, animal infection models and human bodies. The sericin provided by the invention preserves the complete structure and activity of natural sericin, and can enhance the antiviral natural immune response of cells by promoting the activation speed and efficiency of the natural immune response of cells.

The invention is realized by the application of sericin in preparing a medicament for preventing and controlling virus infection.

Further, the sericin is applied to the preparation of medicines for preventing and controlling vesicular stomatitis virus and enterovirus-71 infection.

Further, the concentration of the sericin is 10-20mg/mL, and the effective final concentration of the sericin in a cell culture system or tissue fluid in a cell culture environment/in vivo tissue fluid environment is more than 1 mg/mL.

The extracted protein concentration must be higher than the final concentration after use (cell growth environment). For example: the cell culture system is added with the sericin (10-20mg/mL) with the culture medium volume of 1/10, and the final concentration of the sericin in the cell culture system is 1-2 mg/mL.

In addition, in the case of in vitro cell culture, 1/10, which is added in a larger volume than the volume of the culture medium, may cause cell malnutrition due to excessive dilution of the culture medium.

Even if sericin is administered locally in vivo, the sericin stock solution concentration must be higher than this final concentration in order to achieve a local tissue final concentration of 1 mg/mL.

Furthermore, the content of endotoxin in the sericin solution is lower than 0.25EU/mL, so that the natural immune response signal transduction of cells is not interfered, and the inflammatory reaction is avoided.

Another object of the present invention is to provide a method for extracting sericin, which comprises:

selecting available silkworm cocoons and storage conditions; selecting silkworm cocoons which are subjected to opening breakage within 24 hours after cocooning and are subjected to silkworm pupa removal; storage conditions were as follows: storing selected silkworm cocoon at 4 deg.C or-20 deg.C;

step two, pretreatment and sterilization of silkworm cocoons: shearing the silkworm cocoons into fragments in a sterile environment and placing the fragments in a sterile container; spreading the cut silkworm cocoon fragments on a sterile glass plate in a single-layer dispersion manner, and irradiating in ultraviolet rays; turning over the silkworm cocoon fragments, and irradiating by ultraviolet rays;

dissolving sericin by adopting a lithium bromide method or a urea method;

step four, dialysis and sericin renaturation, namely dialysis of the lithium bromide method product and dialysis of the urea method product in the step three;

step five, concentrating the sericin solution, and concentrating the sericin solution in a sterile environment after the last dialysis is finished;

removing endotoxin in the sericin solution, removing a small amount of endotoxin mixed in the sericin solution by using a protein endotoxin removal kit, and performing operation in a sterile environment;

step seven, filtering the sericin solution: absorbing the sericin solution after removing endotoxin in a sterile environment, and filtering by using a filter;

step eight, detecting and requiring the quality control of the sericin solution, comprising the following steps: measuring sericin content, performing protein electrophoresis, detecting endotoxin, pretreating cells for 12 hours by using the obtained sericin solution at a final concentration of not less than 1mg/mL in an RNA virus infected cell experimental model, and then infecting sericin-treated and non-sericin control cells by using tool virus;

step nine, the sericin solution which completely meets the four detection standards in the step eight is used as a cellular antiviral immunopotentiator for antiviral application.

Further, in the second step, the silkworm cocoons are cut into pieces smaller than 2x 2mm²The pieces of (a) are placed in a sterile container; placing the cut silkworm cocoon fragments in ultraviolet rays with the light intensity of 1400Lux for irradiating for 30 minutes; turning over the silkworm cocoon fragments, and then irradiating the silkworm cocoon fragments for 30 minutes by using ultraviolet rays under the same condition;

in the third step, the sericin dissolution specifically comprises the following steps:

a. lithium bromide method: in a sterile environment, putting each gram of silkworm cocoon fragments into 55mL of sterile lithium bromide aqueous solution with the concentration of 521.1g/L, and oscillating for 6-12 hours at the constant temperature of 37 ℃; placing all the mixture in a sterile centrifuge tube, centrifuging at 12000rpm and 4 ℃ for 15 minutes, taking the supernatant fluid to place in a new centrifuge tube, centrifuging again to take the supernatant fluid, and repeating for 3-5 times until undissolved substances are completely removed;

b. the urea method comprises the following steps: in a sterile environment, putting each gram of silkworm cocoon fragments into 20mL of sterile urea aqueous solution with the concentration of 480g/L, and oscillating for 6-12 hours at the constant temperature of 37 ℃; placing the whole mixture in a sterile centrifuge tube, centrifuging at 12000rpm and 4 ℃ for 15 minutes, taking the supernatant, placing the supernatant in a new centrifuge tube, centrifuging again, taking the supernatant, and repeating for 3-5 times until undissolved substances are completely removed.

Further, in step four, the method for dialyzing the product of the lithium bromide method comprises the following steps: sucking sericin solution, placing in a dialysis bag, sealing, and dialyzing in 10 times volume of sterile deionized water containing 2mM glutathione at 4 deg.C, wherein the 2mM glutathione comprises 1.6mM reducing glutathione GSH and 0.4mM oxidized glutathione GSSG; changing sterile deionized water containing 2mM glutathione once every 6 hours, and dialyzing for 3 times; dialyzing in sterile deionized water without glutathione for 4 times, 6 hours each time;

in the fourth step, the dialysis method of the urea method product comprises the following steps: sucking sericin solution, placing the sericin solution in a dialysis bag, sealing, placing the dialysis bag in 10 times of volume of 6M sterile urea solution containing 2mM glutathione for dialysis for 6 hours, then placing the dialysis bag in 10 times of volume of 2mM glutathione 4M sterile urea solution for dialysis for 6 hours, and then placing the dialysis bag in 10 times of volume of 2mM glutathione 2M sterile urea solution for dialysis for 6 hours; dialyzing in 10 times volume of sterile deionized water without glutathione for 6 hr, periodically replacing sterile deionized water, dialyzing for 4 times with dialysis time not less than 6 hr each time;

step five, completely covering the dialysis bag with polyethylene glycol (PEG) 6000 solid powder, and washing the polyethylene glycol outside the dialysis bag with sterile deionized water after 10 minutes; the volume of the sericin solution concentrated on each gram of silkworm cocoon is 10-20 mL.

Further, step eight includes:

1) measuring the content of sericin: the obtained sericin solution is subjected to concentration determination by using a standard commercial protein concentration detection kit, and the qualified range of the sericin content of the extracted sericin solution is 10-20 mg/mL;

2) protein electrophoresis: taking 10 microliter of the obtained sericin solution, carrying out electrophoresis according to a standard SDS-PAGE protein electrophoresis process, and then carrying out Coomassie brilliant blue staining on the gel to display a protein band; 1 and 2 obvious bands are respectively arranged above and below 250kD of the structurally complete sericin; (as shown in figure 3)

3) Performing endotoxin detection, namely detecting the content of endotoxin in the obtained sericin solution by using a limulus reagent, wherein the content of the endotoxin in the sericin solution which meets the biological safety is lower than 0.25 EU/mL;

4) in the experimental model of RNA virus infected cells, cells were pretreated with the obtained sericin solution at a final concentration of not less than 1mg/mL for 12 hours, and sericin-treated and non-sericin-treated control cells were infected with a tool virus. After detection by virology techniques, the sericin-pretreated experimental group can be seen compared with the control group.

The invention also aims to provide the application of the sericin in the application as a cell natural immune response enhancer in preparing a preventive and therapeutic drug of RNA virus. The sericin dosage concentration is 10-20mg/mL, and the final concentration of cells and tissues is not less than 1mg/mL, so that the effect is optimal.

The invention also aims to provide an immune product prepared by the cell natural immune response enhancer and used for skin and mucous membrane tissues, and an antiviral skin care product and food prepared by the immune product.

Another object of the present invention is to provide a biosafety control system for a sericin solution for carrying out the extraction method of sericin, the biosafety control system for a sericin solution comprising: the device comprises a sterilization and disinfection device for silkworm cocoons and a device for operating the sterilization and disinfection device in a sterile environment, a device for sterilizing articles and reagents, performing sterile operation and dialysis to remove small molecules and endotoxin and a device for filtering and removing large molecules and large particles.

By combining all the technical schemes, the invention has the advantages and positive effects that:

the invention is based on the basic principle of extracting sericin by a urea method, improves the extraction process, and can extract high-purity, high-integrity and non-toxic sericin from the silkworm cocoons. In particular, the sericin product obtained by the method can meet the requirements of sterility, no nuclease and no heat source, and can be used for scientific research of cells and animals and can also be used for human bodies theoretically. The sericin proposed by the invention can be directly used in antiviral tests against various viruses, including cell level and animal level tests. By detecting the infection and replication levels of various viruses under different concentration conditions and different administration modes, the invention finds the necessary conditions for preventing and controlling the viruses and the optimal administration mode of the sericin. Through antiviral tests of different types of viruses, the inventors have clarified the types of viral infectious diseases that sericin can be used to control.

Compared with the existing sericin extraction technology, the method has the following outstanding advantages:

(1) the sericin product obtained by the method has higher purity, higher biological safety and wider application range. The invention meets the requirements of sterilization and aseptic environment of natural silkworm cocoons and aseptic operation standard to ensure the biological safety of sericin in the extraction process, and in addition, adopts an endotoxin removing technology to remove bacterial cell wall pyrogen components possibly mixed in the environment by raw materials, thereby further ensuring the biological safety of the invention. Antiviral agents, if mixed with pyrogens, can severely interfere with the recognition of specific viral components (also the viral pyrogens), affect the cells to initiate the innate immune response, and cause unwanted inflammatory responses. The sericin product provided by the invention can be used for cell culture systems, animal models and human bodies. Taking medical sodium chloride injection (injection is the most strict in requirements of various medical supplies) as an example, the endotoxin standard is less than 0.5EU/mL, and the endotoxin of the sericin solution provided by the invention is less than 0.5 EU/mL. (as shown in figures 4 and 5).

(2) The invention confirms technical parameters such as sericin dissolving, dialyzing and concentrating conditions, liquid volume range and the like through repeated experiments, reduces the occurrence of irreversible sericin precipitation and improves the yield of soluble sericin protein. According to the invention, 10-20mL of 10-20mg/mL sericin solution product (shown in figure 2) can be obtained per 1g of silkworm cocoon, namely 0.1-0.4g (lower limit, minimum volume X minimum concentration, upper limit, maximum volume X maximum concentration) of sericin in a dissolved state can be obtained per 1g of silkworm cocoon theoretically, the concentration is closer to the upper limit when the product volume is the minimum value in actual practice, and 0.2-0.3g of sericin in a dissolved state can be obtained per 1g of silkworm cocoon in actual operation. In fact, the sericin content of 1g of natural silkworm cocoon is about 0.3g, so the yield of the sericin of the invention is between 60 and 100 percent. The technical parameters of sericin dissolution, dialysis and concentration conditions, liquid volume range and the like in the actual operation process are strictly controlled, and the yield can reach more than 90 percent. The existing sericin extraction technology has a plurality of problems of hydrolysis, irreversible precipitation and the like, and the yield of soluble sericin is extremely low.

(3) The invention protects the natural biological activity of sericin to the utmost extent through the structural integrity and the quality control of biological activity.

The sericin which has no pyrogen, complete structure and biological activity and is provided by the invention for the first time can be used as a cellular antiviral immunopotentiator.

The sericin provided by the invention can inhibit intracellular lactic acid production by improving the metabolic state of cells and reducing the level of anaerobic respiration (glycolysis) of the cells. The increase of intracellular lactic acid level can reduce the stability of RIG-I protein of virus RNA receptor, so that the interferon activation pathway of virus RNA/RIG-1/MAVS is inhibited, thereby leading the cell antiviral natural immunity to be in an inhibition state. The RIG-1/MAVS signal level of the cells treated by the sericin is obviously improved, and the sensitivity to virus RNA is greatly improved. (as shown in figure 6)

The sericin provided by the invention can obviously improve the activity of an interferon promoter, and the sericin with the final concentration of 1mg/mL can improve the activity of IFN-promoter by 8-10 times (as shown in figure 7).

The sericin provided by the invention can obviously improve the resistance of cells to vesicular stomatitis virus infection with different infection doses, and the sericin treated cells can completely block the virus infection (as shown in figure 8) when the virus is infected with low dose. The sericin-treated cells provided by the present invention significantly decreased the amount of newly produced virus after infection with vesicular stomatitis virus (as shown in FIG. 9).

The sericin provided by the invention can activate the expression of interferon in a cell in a shorter time and greatly improve the expression level of the interferon when enterovirus-71 (EV71) is infected (as shown in figure 11). The sericin-treated cells provided by the present invention significantly reduced the number of newly produced viral copies after infection with enterovirus-71 (see FIG. 11).

The sericin provided by the invention is a natural protein molecule extracted according with virology requirements, and can be used for preventing and controlling infection of vesicular stomatitis virus and enterovirus-71. The invention can play a role in improving the level of RIG-I/MAVS signals, wherein RIG-I is a recognition element of double-stranded RNA in cells, so that the invention can obviously improve the natural immune response of cells to RNA virus infection and is a cellular immune enhancer aiming at RNA virus infection. (see figures 6, 7, 8, 9, 10, 11).

The invention develops and utilizes the fibroin sericin for the first time, and the fibroin sericin is used for preventing and controlling the virus infection.

Compared with the existing antiviral drugs or technical means, the sericin provided by the invention has the irreplaceable advantages. First, the raw materials are widely available and the application cost is low. The silk cocoon is a raw material for producing silk, is a main product in the mulberry silkworm breeding industry, extracts and produces the antiviral sericin product by utilizing the silk cocoon, and has absolute cost advantage in conversion and application compared with the high synthesis and production cost of other antiviral medicaments. Secondly, the biological safety is higher, and the application range is wider. Sericin as a natural component of the silkworm cocoon has small-scale practical application in skin care products and foods, and the non-toxic and harmless property of the material is effectively proved by practice, so that the technology provided by the invention can be developed into a protective virus product for daily use.

The sericin solution provided by the invention has the effective concentration of not less than 0.1mg/mL and the optimal final effect concentration of not less than 1mg/mL for improving the cell antiviral natural immune response, can be developed into an immunopotentiator for tissues such as skin, mucous membrane and the like, and can also be developed into antiviral products in the forms of skin care products, food and the like.

The invention expands the application of the sericin and develops the potential application value of the sericin. Sericin is treated as waste or cheap by-product in the production step of silk cocoon spinning in silk textile industry, the technology provided by the invention can be integrated and complemented with the traditional silk industry, changes waste into valuable, develops sericin into cellular antiviral immunopotentiator, and provides guarantee for human health.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings needed to be used in the embodiments of the present application will be briefly described below, and it is apparent that the drawings described below are only some embodiments of the present application.

FIG. 1 shows a technical scheme for extracting sericin according to an embodiment of the present invention. A. Silkworm cocoon, silk mimic diagram and silk cross section schematic diagram. The schematic cross section of silk shows that the silk is composed of fibroin at the core and sericin wrapped at the periphery. B. The invention provides a technical flow chart for extracting sericin.

Fig. 2 is the concentration/content control data for the sericin extraction process provided by the embodiment of the invention. In the process of the sericin extraction technology provided by the invention, sericin concentration detection and control data of products obtained in the steps of dissolution, dialysis/renaturation, concentration and the like and final products are obtained. The sericin solution obtained by the two dissolving methods avoids irreversible precipitation of sericin to ensure the yield and obtains a final product with appropriate concentration of sericin by accurately controlling the volume, the concentration and the sericin dissolving limit (the required range is 10-20mg/mL, and the two methods in the embodiment are both controlled to be about 15 mg/mL).

FIG. 3 shows the result of Coomassie blue staining after SDS-PAGE of the sericin solution provided in the example of the invention. The molecular weight of the first band of the protein Ladder from top to bottom is 250kD, and the molecular weight of the second band is 90kD.

FIG. 4 shows the results of an experiment for detecting endotoxin in a sericin solution (LiBr assay) using a limulus reagent according to an example of the present invention. (limulus reagent principle: the higher the endotoxin content, the more gel-forming.

FIG. 5 shows the results of an experiment for detecting endotoxin in a sericin solution (urea method) using a limulus reagent according to an embodiment of the present invention.

FIG. 6 is a graph showing the levels of RIG-I, MAVS, TLR4 and MyD88 in sericin-treated cells and normal culture RD cells (control group) as provided in the examples of the present invention.

FIG. 7 shows the effect of sericin on the promotion of the transcriptional activity of an interferon promoter according to an embodiment of the present invention. The sericin provided by the invention is used for treating HEK293T cells for 24 hours respectively at final concentrations of 0.1mg/mL and 1mg/mL, then the treated and untreated normal cultured cells are transfected with interferon promoter plasmids and Poly (I: C), and luciferase activity is measured after 24 hours of transfection.

FIG. 8 is a graph of inhibition of viral infection by sericin-treated cells provided in an embodiment of the invention. Vesicular Stomatitis Virus (VSV) of different multiplicity of infection (MOI) infected human HepG2 cells for 12 hours, and bright field and fluorescence photographs under a microscope. The viral infection and viral integration of green fluorescent protein (EGFP) was significantly lower in sericin treated cells than in control cells.

FIG. 9 is a graph showing the level of VSV viral replication inhibited in sericin-treated cells as provided in the example. HepG2 cells were incubated for 24 hours with the sericin solution of the present invention at a final concentration of 1 mg/mL. Viral copy number assay results in cell culture supernatants 24 hours after infection of sericin-treated HepG2 cells and normal control cells with Vesicular Stomatitis Virus (VSV) at multiplicity of infection (MOI) of 1.

FIG. 10 shows that the interferon is activated more rapidly and expressed at a higher level when infected with a virus in sericin-treated cells provided in examples of the present invention. RD cells were incubated with the sericin solution of the present invention at a final concentration of 1mg/mL for 24 hours, and treated cells and normal-cultured RD cells were collected as 0-hour samples. Human enterovirus 71(EV71) with multiplicity of infection (MOI) of 1 infected sericin-treated RD cells and normal control cells, and after infection for 3 hours, 6 hours, 10 hours and 14 hours, cell samples were collected, and changes in interferon- α (A), interferon- (B) and interferon- λ 1(C) messenger RNA levels were detected and calculated.

FIG. 11 shows inhibition of the level of EV71 viral replication in sericin-treated cells as provided in the examples. RD cells were incubated with a sericin solution of the present invention at a final concentration of 1mg/mL for 24 hours. Viral copy number assay results in cell culture supernatants 24 hours after infection of sericin-treated RD cells and normal control cells with human enterovirus 71(EV71) with multiplicity of infection (MOI) of 1.

FIG. 12 is a schematic diagram of the design of the sericin extraction technical scheme provided by the embodiment of the invention, closely combined with the requirements of product quality detection and control.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

Aiming at the problems in the prior art, the invention provides sericin, an extraction method, a cellular antiviral immunopotentiator and application thereof, and the invention is described in detail with reference to the accompanying drawings.

The invention provides a sericin extraction technical method which has no pyrogen, high purity, complete structure and biological activity and can meet the research requirements and application requirements of virus disciplines.

The invention provides a combined application scheme of sericin raw material sterilization, an aseptic control flow of extraction, a pyrogen removing technology, a technology for ensuring the dissolution, renaturation, concentration, purification and the like of a sericin complete protein structure.

The invention can meet the detection and quality control technology and standard of sericin researched and applied by virus discipline. Namely: protein concentration determination (in the range of 10-20mg/mL sericin concentration) + SDS-PAGE electrophoresis (three visible bands) + pyrogen detection (endotoxin content less than 0.25EU/mL) + biological activity detection (RNA virus infection and replication inhibition level).

The invention relates to an operation and control system which closely combines the quality control requirement of sericin and an extraction process.

The invention provides a biological safety control system for producing sericin solution, which is used for sterilizing and disinfecting silkworm cocoons, sterilizing the silkworm cocoons, performing aseptic articles and reagents, performing aseptic operation, removing micromolecules through dialysis, removing endotoxin, filtering and removing macromolecules and large particles.

The invention provides a method for sterilizing and disinfecting silkworm cocoons, which comprises the following steps: shearing into pieces smaller than 2 × 2mm under sterile environment²The pieces of (4) were spread on a sterile glass plate in a single layer, irradiated with ultraviolet rays having a light intensity of 1400Lux for 30 minutes, and then turned over, irradiated with ultraviolet rays under the same conditions for another 30 minutes.

The invention provides a sericin lithium bromide solution dialysis renaturation method which comprises the following steps: in a sterile environment at 4 ℃, sucking the sericin solution, placing the sericin solution in a dialysis bag (MW3500) for sealing, placing the dialysis bag in 10 times of volume of sterile deionized water containing 2mM glutathione (including 1.6mM reduced glutathione GSH and 0.4mM oxidized glutathione GSSG) for dialysis, replacing the sterile deionized water containing 2mM glutathione once every 6 hours, and dialyzing for 3 times. Followed by dialysis against sterile deionized water (without glutathione) 4 more times for 6 hours each.

The invention provides a sericin urea solution dialysis renaturation method which comprises the following steps: in a sterile environment at 4 ℃, sucking the sericin solution, placing the sericin solution in a dialysis bag (MW3500) for sealing, placing the solution in 10 times of volume of 6M sterile urea solution (containing 2mM glutathione) for dialysis for 6 hours, then placing the solution in 10 times of volume of 4M sterile urea solution (containing 2mM glutathione) for dialysis for 6 hours, and then placing the solution in 10 times of volume of 2M sterile urea solution (containing 2mM glutathione) for dialysis for 6 hours. Then placing the mixture into 10 times of sterile deionized water (without glutathione) for dialysis for 6 hours, periodically replacing the sterile deionized water, and dialyzing for 4 times, wherein the dialysis time is not less than 6 hours each time.

The invention provides a modified sericin renaturation liquid formula which comprises the following components in parts by weight: use of sterile deionised water containing 1.6mM reduced Glutathione (GSH) and 0.4mM oxidised glutathione (GSSG) for sericin renaturation.

The invention provides an application of a 6M sterile urea solution containing 1.6mM of reducing Glutathione (GSH) and 0.4mM of oxidized glutathione (GSSG) in sericin renaturation.

The invention provides an application of a 4M sterile urea solution containing 1.6mM of reducing Glutathione (GSH) and 0.4mM of oxidized glutathione (GSSG) in sericin renaturation.

The invention provides an application of a 2M sterile urea solution containing 1.6mM of reducing Glutathione (GSH) and 0.4mM of oxidized glutathione (GSSG) in sericin renaturation.

The invention provides a sericin dissolving and concentrating content/concentration control system which comprises the following components: dissolving 55mL of 6M lithium bromide or 20mL of 8M urea in every 1g of silkworm cocoon, controlling the volume of the sericin solution corresponding to 1g of silkworm cocoon to be 10-20mL during concentration, and controlling the concentration of the sericin protein of the product which can be used for antivirus application to be 10-20 mg/mL.

The invention provides a sericin complete structure and biological activity control method, which comprises the following steps: selecting and storing (storing silkworm cocoon after silkworm cocoons are cracked and silkworm pupas are removed within 24 hours at 4 ℃ or-20 ℃), and performing denaturation and dissolution.

The sericin provided by the invention is used for preventing and controlling vesicular stomatitis virus infection.

The sericin provided by the invention is used for preventing and controlling enterovirus-71 infection.

The sericin provided by the invention can be used as a cell natural immune response enhancer and applied to the prevention and treatment of RNA viruses.

The sericin provided by the invention can be used as an auxiliary material or an additive for preparing medicines and medical supplies for preventing and treating virus infectious diseases.

The sericin provided by the invention is used for preparing daily supplies such as food, skin care products, disinfectants, detergents and the like, and is used as an additive component to achieve the effect of preventing virus infection.

The present invention is further described below in conjunction with a sericin extraction protocol that can be used for antiviral.

As shown in fig. 1, the method for extracting sericin for resisting viruses provided by the invention comprises the following steps:

(1) selecting available silkworm cocoons and storage conditions. The silkworm cocoon selection method comprises the following steps: selecting silkworm cocoons which are subjected to opening breakage within 24 hours after cocooning and are removed from silkworm pupas. The sericin component of the silk can be gradually damaged when the silkworm chrysalis is not removed from the cocoons after cocooning, and the application effect of the silk can be gradually reduced when the silk fibroin composition is used for the silk fibroin material. Storage conditions were as follows: the selected silkworm cocoon can be stored at 4 deg.C or-20 deg.C for at least one year.

(2) Pretreating and sterilizing silkworm cocoons: in sterile environment (in biological safety cabinet or clean bench), cutting Bombyx Bombycis into pieces smaller than 2x 2mm with sterilized tweezers and scissors²The pieces of (a) are placed in a sterile container. The cut pieces of silkworm cocoon are spread on a sterile glass plate in a single layer and irradiated for 30 minutes in ultraviolet light of 1400 Lux. After the silkworm cocoon fragments are turned over, the ultraviolet rays are irradiated for 30 minutes under the same condition.

(3) Dissolving sericin: the method comprises the following steps:

a. lithium bromide method. In a sterile environment, throwing each gram of silkworm cocoon fragments into 55mL of sterile lithium bromide (LiBr) water solution with the concentration of 521.1g/L (6M), and oscillating at the constant temperature of 37 ℃ for 6-12 hours. Then, the whole mixture was put into a sterile centrifuge tube, centrifuged at 12000rpm at low temperature (4 ℃) for 15 minutes, the supernatant was taken into a new centrifuge tube, centrifuged again to take the supernatant, and repeated 3-5 times until the undissolved substances were completely removed.

b. Urea process. In a sterile environment, throwing each gram of silkworm cocoon fragments into 20mL of sterile urea aqueous solution with the concentration of 480g/L (8M), and oscillating for 6-12 hours at the constant temperature of 37 ℃. Then, the whole mixture was put into a sterile centrifuge tube, centrifuged at 12000rpm at low temperature (4 ℃) for 15 minutes, the supernatant was taken into a new centrifuge tube, centrifuged again to take the supernatant, and repeated 3-5 times until the undissolved substances were completely removed.

(4) Dialysis and renaturation of sericin: step a in step (3), dialyzing the product of the lithium bromide method: in a sterile environment at 4 ℃, sucking the sericin solution, placing the sericin solution in a dialysis bag (MW3500) for sealing, placing the dialysis bag in 10 times of volume of sterile deionized water containing 2mM glutathione (including 1.6mM reduced glutathione GSH and 0.4mM oxidized glutathione GSSG) for dialysis, replacing the sterile deionized water containing 2mM glutathione once every 6 hours, and dialyzing for 3 times. Followed by dialysis against sterile deionized water (without glutathione) 4 more times for 6 hours each.

Step (3), dialysis of the product of the urea method in step b: in a sterile environment at 4 ℃, sucking the sericin solution, placing the sericin solution in a dialysis bag (MW3500) for sealing, placing the solution in 10 times of volume of 6M sterile urea solution (containing 2mM glutathione) for dialysis for 6 hours, then placing the solution in 10 times of volume of 4M sterile urea solution (containing 2mM glutathione) for dialysis for 6 hours, and then placing the solution in 10 times of volume of 2M sterile urea solution (containing 2mM glutathione) for dialysis for 6 hours. Then placing the mixture into 10 times of sterile deionized water (without glutathione) for dialysis for 6 hours, periodically replacing the sterile deionized water, and dialyzing for 4 times, wherein the dialysis time is not less than 6 hours each time.

(5) Concentrating the sericin solution: after the last dialysis, the dialysis bag filled with the sericin solution is horizontally placed in a sterile plate under a sterile environment, polyethylene glycol (PEG6000) solid powder is taken to completely cover the dialysis bag, and after 10 minutes, the polyethylene glycol outside the dialysis bag is washed clean by sterile deionized water. Ensuring that the volume of the sericin solution concentrated on each gram of silkworm cocoon is 10-20mL, and if the volume is larger than the volume, repeating the concentration process.

(6) Removing endotoxin in the sericin solution: the invention adopts a protein endotoxin removing kit to thoroughly remove a small amount of endotoxin possibly mixed in a sericin solution, and the operation is carried out in a sterile environment.

(7) Filtering the sericin solution: the endotoxin-cleared sericin solution was aspirated by a syringe under a sterile environment and filtered once through a filter having a pore size of 0.22 μm.

(8) Sericin solution quality control detection and requirements include:

1) measuring the content of sericin: the obtained sericin solution is subjected to concentration determination by using a standard commercial BCA protein concentration detection kit, and the qualified range of the sericin content of the extracted sericin solution is 10mg-20 mg/mL.

2) Protein electrophoresis: a small amount (about 10. mu.l) of the obtained sericin solution was subjected to electrophoresis according to the standard SDS-PAGE protein electrophoresis procedure, and then Coomassie brilliant blue staining was performed on the gel to show protein bands. Structurally intact sericin has 1 and 2 distinct bands above and below 250kD, respectively.

3) And (3) endotoxin detection, namely detecting the content of endotoxin in the obtained sericin solution by utilizing a commercial limulus reagent, wherein the content of the endotoxin in the sericin solution which meets the biological safety is lower than 0.25 EU/mL.

4) In an experimental model of RNA virus-infected cells, cells were pretreated with the obtained sericin solution at a final concentration of not less than 1mg/mL for 12 hours, after which sericin-treated and non-sericin-treated control cells were infected with a tool virus (e.g., VSV-EGFP). After detection by virology technology, the experimental group pretreated by sericin can be seen to have obviously reduced virus infection and replication level and obviously reduced cell apoptosis caused by virus infection compared with a control group.

(9) The sericin solution which completely meets the four detection standards in the step (8) can be used as a cellular antiviral immunopotentiator for experimental research and antiviral application. The technical scheme provided by the invention can ensure that the technical method, the flow and the parameters meet various standards of sericin solution quality control detection (step (8)).

In step (8), the design of the sericin extraction technical scheme provided by the invention is tightly combined with the requirements of product quality detection and control, as shown in fig. 12, comprising

The invention provides a technical scheme for extracting sericin with high biological safety, complete structure, high purity and biological activity for the first time, and sericin which can be used for cell virus infection experimental models, animal virus infection models and human body antiviral infection applications is extracted according to the technical requirements of virology.

Controlling the content/concentration of sericin in the product. Based on the results of experiments on the induction of interferon by sericin at different concentrations (shown in FIG. 7), the inventors have clarified that the final concentration of sericin for promoting natural immune response against virus of cells should not be less than 1mg/mL because the volume of the culture medium should be considered in the in vitro cell culture system if the extracted sericin is applied to virus resistance, and the administration volume and concentration should be considered more if it is applied to human body, so that the concentration of the extracted sericin solution is required to be at least 10 times the effective final concentration, i.e., 10 mg/mL. If the concentration of the sericin product is lower than 10mg/mL, if antiviral experiments are carried out, the sericin liquid required to be added into a cell culture system is too large, so that the culture medium is excessively diluted, the nutrition and the state of cells are influenced, and the antiviral effect cannot be realized. Therefore, the sericin content of the sericin solution which meets the requirement must be higher than 10 mg/mL. In addition, the sericin solution can not be concentrated by the traditional protein concentration means, and the concentration of the sericin can be improved by concentrating by a freeze-drying method. The process of freezing and thawing the sericin solution or precipitating and separating out sericin is not reversible, namely, the sericin is difficult to dissolve once separated out in a precipitate form, and the dissolution rate of completely lyophilized sericin is very low and far less than 10 mg/mL. The inventors have found that irreversible precipitation occurs at room temperature at neutral pH when the concentration of sericin is higher than 20mg/mL, when the concentration is concentrated by reducing the solvent (water) of the sericin solution at room temperature. Finally, based on the special solubility of the biological material and a large number of research test results of the inventor, the concentration range of the sericin solution for antivirus application is defined in the range of 10-20mg/mL, and the volume of the sericin solution during concentration is controlled in the range of 10-20mL corresponding to 1g of silkworm cocoon.

The technical scheme provided by the invention adopts the following three technical measures to ensure the concentration of the sericin protein: (1) in the step of selecting and storing the silkworm cocoons, the inventor selects fresh silkworm cocoons and stores the silkworm cocoons at a low temperature, so that sericin in raw materials is well ensured not to be degraded in the environment, and the content of the sericin in the raw materials is ensured to the maximum extent; (2) in the step of dissolving sericin, the inventor selects a LiBr and urea dissolving method, and compared with a thermal dissolving and alkali dissolving method, the degradation of sericin in the dissolving process is reduced; (3) in the sericin concentration step, after the dialysis is directly finished, the dialysis bag filled with the sericin solution is directly contacted with the polyethylene glycol solid to separate out water in the dialysis bag, so that the purpose of sericin concentration is achieved, the volume of liquid in the dialysis bag is closely related during concentration, the concentration of sericin with excessive residual volume is insufficient, and sericin with too small volume can be irreversibly separated out. The inventors confirmed that the concentration of the sericin solution per 1g of silkworm cocoon was completed in a volume range of 10 to 20mL by repeating the tests for a plurality of times. The immediate volume of the sericin solution can be estimated by weighing the total weight of the dialysis bag. The invention obtains sericin products with the content required by antiviral application (as shown in attached figures 2, 6, 7, 8, 9, 10 and 11) by the technical control of the three extraction processes.

Control of sericin structural integrity. The structural integrity of the protein molecule determines the functional integrity, and complete sericin molecules can be obtained, which is a precondition for researching and utilizing the biological activity of the sericin molecules. The invention ensures the complete structure of the extracted sericin protein (as shown in the attached figure III), and adopts the following technical means: (1) during the process of selecting and storing the raw materials, sericin is protected from being naturally degraded to the maximum extent so as to damage the structure. In a natural state, the sericin on the silkworm cocoon not only gradually reduces the total content, but also gradually reduces the molecular integrity of the sericin during the process that the silkworm finishes cocooning and finishes metamorphosis development and grows into a moth and breaks the cocoon. The damage of sericin molecules is related to the exposure to the natural environment, and is more closely related to the utilization of sericin in the development process of silkworms. According to the technical scheme provided by the invention, silkworm pupas are broken and removed at the first time of silkworm cocoon formation, so that the structural integrity of sericin is effectively protected. (2) The adopted dissolving method avoids the integrity of sericin molecules from being damaged by high temperature or alkali. (3) The time required for the dialysis and renaturation steps is the longest in the whole technical process, and in order to reduce the possibility that sericin molecules are hydrolyzed or degraded by protease in the long-term dialysis process, the dialysis process is selected to be carried out at a temperature of 4 ℃.

Purity and biosafety control. Sericin for antiviral applications, whether used for antiviral tests at the cellular level or antiviral applications at the individual level, must have high requirements for purity and biosafety. Firstly, the extracted sericin cannot contain excessive impurities, although the silkworm cocoon mainly consists of silk and the silk only consists of two proteins of fibroin and sericin, the impurities possibly mixed in the environment, the possibility of mixing the impurities and microorganisms in each process of the rough extraction method, and the sericin with low purity cannot meet the requirement of the application biosafety of the invention. Secondly, if the extracted sericin contains a certain impurity component, it interferes with or inhibits the biological activity of sericin, and it is not allowed to contain even if its content is low. Therefore, the control of purity and biosafety is a precondition and basis for the development and application of sericin biological activity and related functions thereof.

The invention ensures the safety of sericin product extraction (as shown in the attached figures 4 and 5), and is designed by the following technical scheme: (1) the whole extraction process is carried out in an aseptic environment, all used instruments are sterilized, all used reagents are autoclaved or sterilized by filtration, and the operation process completely complies with the requirements of aseptic operation experiments. (2) The cut pieces of silkworm cocoon must be subjected to aseptic processing before dissolution because dissolution is carried out at 37 ℃ and for a long time, a small amount of microorganisms mixed in the raw material of the silkworm cocoon may be proliferated in the dissolution system in a large amount. The silkworm cocoon fragments can not be sterilized at high temperature and high pressure (the sericin can be damaged) or by chemical drugs (impurities can be mixed), the inventor adopts an ultraviolet sterilization method to disperse and spread the silkworm cocoon fragments on a sterile glass plate in a single layer, and the silkworm cocoon fragments are irradiated in ultraviolet rays with the light intensity of 1400Lux for 30 minutes. After the silkworm cocoon fragments are turned over, the ultraviolet rays are irradiated for 30 minutes under the same condition. The ultraviolet intensity and the irradiation time can ensure that bacteria or fungal spores possibly adsorbed on the silkworm cocoon fragments can be completely killed, and the molecular structure of sericin can not be damaged. (3) After the dissolution is completed, LiBr and urea used for the dissolution must be removed from the sericin solution, and since they are both small molecular compounds, a dialysis method is used. After the LiBr solution (LiBr original concentration is 6M) dissolved with sericin is fully dialyzed by 10 times of volume of sterile deionized water for 7 times, because the small-molecule LiBr can freely diffuse at the outer side in the dialysis bag, the LiBr concentration in the sericin solution is theoretically obtained after 7 times of dialysis<6x 10^-7M, namely 0.0000521.1g of sericin solution per liter, LiBr of the concentration is completely within the biological safety range, and the sericin extracted by the LiBr method is only used in cell experiments and animals and not used in human bodies. The urea solution dissolved with sericin is dialyzed in 10 times volume of sterile deionized water for 4 times after the concentration of urea is reduced to 2M, and theoretically, the concentration of urea in the sericin solution is finally obtained<2x 10^-4M, i.e., 0.012g urea per liter of sericin solution, which is also within the biosafety range. (4) And the protein endotoxin removal kit is adopted to carry out endotoxin removal operation on sericin, so that a small amount of endotoxin which can be adsorbed by the raw material is thoroughly removed, and the biological safety of the product is further ensured. (5) Purifying the sericin solution by using a filter with the pore diameter of 0.22 micron to remove possible mixed supermolecule, large-particle impurities or microorganisms, and further ensuring the purity and biological safety of the sericin.

And controlling the biological activity of sericin. (1) The selection and preservation measures of silkworm cocoons are the first step of ensuring the biological activity of sericin and are important. (2) The invention adopts LiBr and urea to carry out conformation denaturation on sericin (to destroy the three-level and four-level structures of sericin) for dissolving, then a sericin solution dialysis bag containing the denatured and dissolved sericin is put into sterile deionized water containing a renaturation agent for low-temperature dialysis renaturation, the denatured sericin is dissolved and dissolved in a high-concentration LiBr or urea solution in a form of polypeptide, a dialysis system is adopted for gradually reducing the concentration of LiBr or urea, the polypeptide chain of sericin can be gradually folded, the combination of disulfide bonds can not naturally correspond to the corresponding sulfydryl (-SH) in natural sericin in the refolding process, and the mismatching can be generated, renaturation agents (2mM glutathione, including 1.6mM reduced glutathione GSH and 0.4mM oxidized glutathione GSSG) are added into the system to maintain the reduction conditions of the solution, so as to allow 'mispairing' and finally tend to be thermodynamically more stable pairing, and the polypeptide chain of the protofilament glue protein is spontaneously folded into a natural conformation. This step is a key step in ensuring the activity of native sericin.

Those of ordinary skill in the art of the methods provided herein may also perform other steps, and the invention of fig. 1 is provided as a specific example only.

The invention is further described with reference to specific examples.

Example 1:

as shown in FIG. 1, the complete structure and high biological safety can be used for sericin extraction for antiviral application (LiBr method)

(1) Selecting 1g of silkworm cocoon, and then removing silkworm pupa cocoon within 24 hours, or selecting silkworm cocoon which is stored at 4 ℃ and meets the condition.

(2) Pretreating and sterilizing the silkworm cocoon. In a biological safety cabinet (Thermo), silkworm cocoons are cut into pieces smaller than 2x 2mm with sterilized tweezers and scissors²The pieces of (4) were spread in a single layer on a sterile glass plate and irradiated with ultraviolet light of 1400Lux for 30 minutes. After the silkworm cocoon fragments are turned over, the ultraviolet rays are irradiated for 30 minutes under the same condition.

(3) In a biosafety secondary laboratory, each gram of silkworm cocoon fragments are put into 55mL of sterile lithium bromide (LiBr, Chinese medicine) water solution with the concentration of 521.1g/L (6M), and the mixture is shaken at the constant temperature of 37 ℃ for 6 hours. Then, the whole mixture was put into a sterile centrifuge tube, centrifuged at 12000rpm at low temperature (4 ℃) for 15 minutes, the supernatant was taken into a new centrifuge tube, centrifuged again to take the supernatant, and repeated 3-5 times until the undissolved substances were completely removed.

(4) In a biosafety secondary laboratory, the lithium bromide-dissolved sericin solution was aspirated and placed in a dialysis bag (MW3500) sealed, placed in 10 volumes of sterile deionized water containing 2mM glutathione (including 1.6mM reduced glutathione GSH and 0.4mM oxidized glutathione GSSG, Sigma), dialyzed in a refrigerator at 4 ℃, replaced with sterile deionized water containing 2mM glutathione (ex PALL laboratory water meter and autoclaved) every 6 hours, and co-dialyzed 3 times. Followed by dialysis against sterile deionized water (without glutathione) 4 more times for 6 hours each.

(5) After the last dialysis, in a biosafety second-level laboratory, the dialysis bag filled with the sericin solution is horizontally placed in a sterile plate, the dialysis bag is completely covered with polyethylene glycol (PEG6000, Chinese medicine) solid powder, and after 10 minutes, the polyethylene glycol outside the dialysis bag is washed clean by sterile deionized water. And (3) weighing and estimating the volume of the sericin solution in the dialysis bag by using an electronic balance, ensuring that the volume of the sericin solution concentrated on each gram of silkworm cocoon is 10-20mL, and if the volume is more than 20mL, repeating the concentration process.

(6) In the biosafety secondary experiment, the sericin solution was thoroughly cleared of endotoxin which may be mixed with the sericin solution using a protein endotoxin removal kit (Cloud-Clone, IS002, according to the product instructions).

(7) In the biosafety secondary experiment, the endotoxin-cleared sericin solution was aspirated by a syringe (manufactured by BD Co.), filtered once with a filter having a pore size of 0.22 μm (manufactured by BD Co.), and the sample was taken in a sterile bag-lid container for use.

Example 2:

the complete structure and high biological safety can be used for sericin extraction (urea method) for antiviral application.

(2) Pretreating and sterilizing the silkworm cocoon. In a biosafety cabinet (Thermo), cocoons were chopped into pieces of less than 2x 2mm 2 using sterilized forceps and scissors, spread in a monolayer on a sterile glass plate, and exposed to ultraviolet light at a light intensity of 1400Lux for 30 minutes. After the silkworm cocoon fragments are turned over, the ultraviolet rays are irradiated for 30 minutes under the same condition.

(3) In a biosafety secondary laboratory, 5 g of silkworm cocoon fragments are put into 100mL of sterile urea (Chinese medicine) aqueous solution with the concentration of 480g/L (8M), and the mixture is shaken at the constant temperature of 37 ℃ for 6 to 12 hours. Then, the whole mixture was put into a sterile centrifuge tube, centrifuged at 12000rpm at low temperature (4 ℃) for 15 minutes, the supernatant was taken into a new centrifuge tube, centrifuged again to take the supernatant, and repeated 3-5 times until the undissolved substances were completely removed.

(4) In a biosafety secondary laboratory, a sericin solution dissolved by urea is placed in a dialysis bag (MW3500) and sealed, and is placed in a 10-fold volume of a 6M sterile urea solution (containing 2mM glutathione) for dialysis for 6 hours, then is placed in a 10-fold volume of a 4M sterile urea solution (containing 2mM glutathione), is dialyzed for 6 hours at 4 ℃, and then is placed in a 10-fold volume of a 2M sterile urea solution (containing 2mM glutathione) for dialysis for 6 hours. Then placing the mixture into 10 times of sterile deionized water (without glutathione) for dialysis for 6 hours, periodically replacing the sterile deionized water, and dialyzing for 4 times, wherein the dialysis time is not less than 6 hours each time.

Example 3:

controlling the quality of the sericin extraction process and detecting the quality of the product sericin solution.

(1) Extracting sericin solution dissolved by LiBr and urea in the process, dialyzing/renaturing product, concentrating product and finally obtaining sericin solution, sucking 10uL in each step, and storing at-20 ℃. After completion of the extraction, a commercial BCA protein concentration detection kit (Milipore) was purchased, and the collected solution was subjected to protein concentration measurement according to the procedures of the kit instructions.

The concentration determination result is shown in figure 2, and by concentration/volume control, irreversible separation of sericin in the extraction process is avoided, so that the yield is ensured, and the concentration of the obtained product sericin solution meets the application requirements of preventing and controlling viruses (shown in figure 2). 16.4mL of final product sericin solution obtained by extracting 1g of silkworm cocoon by a LiBr method has the concentration of 15.6mg/mL and the calculation yield of 85.3 percent. 18.7mL of final product sericin solution obtained by 1g of silkworm cocoon through urea extraction has the concentration of 14.3mg/mL and the calculation yield of 89.1 percent.

Table: sericin extraction product volume, concentration and yield

(2) Taking 10uL of the final product sericin solution, preparing samples according to a standard SDS-PAGE electrophoresis sample preparation method, then loading the samples together with the protein ladder, and starting 110V constant voltage electrophoresis until a bromophenol blue indicator band runs to the bottom of the gel. And taking down the gel, carrying out Coomassie brilliant blue staining and decoloring, observing protein bands of a sericin sample lane in the gel, and photographing the gel.

As shown in figure 3, the sericin solution extracted by the two methods shows three main bands after SDS electrophoresis, which indicates that the product extracted by the method has a more complete natural sericin structure.

(3) A commercial endotoxin extraction kit (limulus reagent) was purchased, 100uL of the sericin solution obtained by extraction was taken for endotoxin detection, and the results of the sericin solution were compared with negative and positive controls. As shown in the attached figures 4 and 5, the endotoxin content of the sericin solution obtained by the two methods is lower than 0.25EU/mL, which indicates that the biological safety of the invention is higher.

(4) Biological activity test, mainly testing the promotion effect of the obtained sericin on the antiviral natural immune response of cells, and referring to example 4 and example 5.

Example 4:

the present invention treated cells are tested against vesicular stomatitis virus

(1) According to the concentration value of the sericin solution of the present invention, the sericin solution was diluted to 10mg/mL with DMEM (Gibico) medium (basic medium for short) containing diabody (Gibico) and fetal bovine serum (Gibico) for use.

(2) Cultured HepG2 cells were plated on a 24-well plate and cultured to a density of 50% (cell coverage plate bottom area ratio), half of the wells were replaced with 0.45mL of fresh basal medium and 0.05mL of 10mg/mL sericin solution of the present invention per well (sericin final concentration of 1mg/mL), and the other half of the wells were replaced with 0.5mL of fresh basal medium as a control, and the culture was continued.

(3) Collecting and detecting virus RNA receptor signal gene. Cells were harvested after 24 hours of culture. The fluorescent quantitative PCR technology is used for detecting the expression levels of genes such as RIG-I, MAVS, TLR4, MyD88 and the like in a cell sample.

As shown in figure 6, the expression level of MAVS in HepG2 cells RIG-I cultured by sericin is obviously higher than that of control cells, which indicates that the sericin provided by the invention can improve the levels of intracellular viral RNA receptor RIG-I and signal transduction molecule MAVS, and has obvious promotion effect on TLR4 and MyD 88.

(4) After an additional 24 hours, all wells were changed to 0.5mL fresh basal medium and I MOI VSV-EGFP virus (2.5X 10 per well) was added⁴Cell count) and gently shaking the medium, removing the virus-containing medium after 2 hours, changing to an equal volume of fresh basal medium, and continuing the culture.

(5) Culturing HEK293T cells in a 24-well plate by adopting sericin with the final concentration of 0.1mg/mL and 1mg/mL for 24 hours by the same method in the step (2), then transfecting the cells treated by two sericins and the cells cultured normally with Poly (I: C) and IFN-promoter plasmids (the ratio of the two is 2: 1), after the transfection is completed for 6 hours, replacing fresh culture medium, culturing for 18 hours, and then detecting luciferase activity.

As shown in figure 7, 0.1mg/mL and 1mg/mL sericin can improve the activity of IFN-promoter, the effect of 1mg/mL sericin is more remarkable, and the activity of IFN-promoter is improved by 8-10 times.

(6) Culturing HepG2 cells in a 24-well plate by the same method as in the step (2) with sericin at a final concentration of 1mg/mL for 24 hours, then infecting with VSV-EGFP viruses at a multiplicity of infection of 10MOI,1MOI,0.1MOI and 0.01MOI, respectively, and after 12 hours, evaluating the virus infection by observing green fluorescence by a fluorescence microscope.

As shown in FIG. 8, the sericin-treated cells showed a significant inhibitory effect on viral infection at different multiplicity of infection, and in the case of viral infection at a minimum multiplicity of infection of 0.01MOI, the sericin-treated cells completely inhibited viral infection.

(7) Culturing cells and normal cells with sericin with the final concentration of 1mg/mL for 24 hours, and after 24 hours of infection by VSV-EGFP with the multiplicity of infection of 1MOI, collecting cell culture supernatant, extracting RNA, and detecting the copy number of virus RNA by a fluorescent quantitative PCR technology.

As shown in FIG. 9, the number of viral copies in the supernatant after infection of normal cells with virus was about 1.5X10⁷Viral copy number per ml of sericin cultured cell supernatant was about 4X10⁵Per ml.

Example 5:

the present invention treated cells are tested against human enterovirus 71

(1) RD cells cultured for 24 hours and RD cells cultured normally are infected by EV71 with the infection multiplicity of 5MOI after culturing with sericin with the final concentration of 1mg/mL, sericin-treated and untreated RD cell samples are collected at 3, 6, 10 and 14 hours before infection and after virus infection, and the expression levels of interferon-alpha, interferon-lambda 1 are detected by a fluorescence quantitative PCR technology after RNA extraction.

As shown in FIG. 10, 3 hours after viral infection, interferon- α, interferon-and interferon- λ 1 were activated in sericin-cultured cells, and RNA expression reached maximum, and activation rate and expression were significantly higher than those in cells not cultured with sericin.

(2) Culturing RD cells with a final concentration of 1mg/mL sericin for 24 hours and normally cultured RD cells, infecting by EV71 with the multiplicity of infection of 5MOI, collecting sericin-treated and untreated RD cell supernatants 24 hours after virus infection, extracting RNA, and detecting the copy number of virus RNA by a fluorescent quantitative PCR technology.

As shown in FIG. 11, the number of viral copies in the supernatant was approximately 1.75X10 24 hours after EV71 infection of normally cultured RD cells⁷ Viral copy number 5 × 10 in cell supernatants cultured on sericin⁵。

The above description is only for the purpose of illustrating the present invention and the appended claims are not to be construed as limiting the scope of the invention, which is intended to cover all modifications, equivalents and improvements that are within the spirit and scope of the invention as defined by the appended claims.

Claims

1. Use of sericin in the preparation of a medicament for preventing and controlling viral infection.

2. Use according to claim 1, wherein the sericin is used for preparing a medicament for preventing and controlling vesicular stomatitis virus, enterovirus-71 infection.

3. The use according to claim 1, wherein the sericin is extracted at a concentration of 10-20mg/mL, and the effective final concentration of sericin in a cell culture system or a tissue fluid in a cell culture environment/in vivo tissue fluid environment is >1 mg/mL.

4. A method for extracting sericin for use as defined in any one of claims 1 to 3, wherein the method for extracting sericin comprises:

dissolving sericin by adopting a lithium bromide method or a urea method;

5. The method for extracting sericin according to claim 4, wherein in the second step, the silkworm cocoon is cut into pieces smaller than 2x 2mm²The pieces of (a) are placed in a sterile container; placing the cut silkworm cocoon fragments in ultraviolet rays with the light intensity of 1400Lux for irradiating for 30 minutes; turning over the silkworm cocoon fragments, and then irradiating the silkworm cocoon fragments for 30 minutes by using ultraviolet rays under the same condition;

6. The method for extracting sericin according to claim 4, wherein in the fourth step, the dialysis of the product of the lithium bromide method comprises: sucking sericin solution, placing in a dialysis bag, sealing, and dialyzing in 10 times volume of sterile deionized water containing 2mM glutathione at 4 deg.C, wherein the 2mM glutathione comprises 1.6mM reducing glutathione GSH and 0.4mM oxidized glutathione GSSG; changing sterile deionized water containing 2mM glutathione once every 6 hours, and dialyzing for 3 times; dialyzing in sterile deionized water without glutathione for 4 times, 6 hours each time;

7. The method for extracting sericin according to claim 4, wherein the eighth step comprises:

1) measuring the content of sericin: the obtained sericin solution is subjected to concentration determination by using a standard commercial protein concentration detection kit, and the qualified range of the sericin content of the extracted sericin solution is 10mg-20 mg/mL;

2) protein electrophoresis: taking 10 microliter of the obtained sericin solution, carrying out electrophoresis according to a standard SDS-PAGE protein electrophoresis process, and then carrying out Coomassie brilliant blue staining on the gel to display a protein band; 1 and 2 obvious bands are respectively arranged above and below 250kD of the structurally complete sericin;

8. Use of sericin as a cellular natural immune response enhancer in the preparation of a preventive and therapeutic drug for RNA virus at a local concentration of not less than 1mg/mL for the use according to claims 1 to 3.

9. An immune product, an antiviral skin care product and a food for skin and mucosal tissue prepared by using the natural cellular immune response enhancer in the application of claim 8.

10. A biosafety control system for a sericin solution for carrying out the extraction method of sericin according to any one of claims 4 to 7, comprising: the device comprises a sterilization and disinfection device for silkworm cocoons and a device for operating the sterilization and disinfection device in a sterile environment, a device for sterilizing articles and reagents, performing sterile operation and dialysis to remove small molecules and endotoxin and a device for filtering and removing large molecules and large particles.